Prostate cancer affects 1 in 6 men and is the second leading cause of cancer death. Definitive diagnosis is based on systematic transrectal ultrasound (TRUS)-guided prostate biopsy typically prompted by the widespread use of prostate specific antigen (PSA) screening. However, this procedure misses about 40% of prostate cancers leading to repeat biopsies at significant patient morbidity and healthcare cost. Therefore, there is an unmet critical need for image-guided biopsy that maximizes the probability of detection of clinically relevant tumors in order to circumvent the current underdiagnsois (and undertreatment) of lethal tumors and conversely overdiagnosis (and overtreatment) of indolent tumors. Image-guided prostate tumor detection and characterization will pave the way for rational treatment management strategy including the emerging focal therapy for organ-confined high grade tumors. Multiparametric magnetic resonance imaging (mpMRI) has been reported to offer relatively high specificity but variable sensitivity for prostat cancer detection. The potential use of positron emission tomography (PET) with a variety of tracers has primarily focused on the biochemical recurrence and the castrate-resistant metastatic phases of the disease. There is currently paucity of data for any PET tracer relevant to the accurate detection, localization, and characterization of primary tumor in patients with suspected prostate cancer, particularly after an initial negative standard TRUS guided biopsy. We have had a long record of interest and experience with the synthesis and evaluation of the cellular proliferation and cellular stress biomarker, 18F-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (18F-FMAU) in conjunction with PET. Our efforts have included completion of an R21-funded preclinical evaluation of 18F-FMAU in animal models of prostate cancer, and since the prior review, attainment of sufficient funds for an IRB-RDRC approved proof-of-concept clinical case study in one patient, and recent submission of an Investigational New Drug application to the US Food and Drug Administration. We are now in a strategic position to continue along this line of research with additional pilot human-based feasibility tria. Therefore, the objective of this revised R21 proposal is to perform a systematic study of 18F-FMAU in men with suspected primary prostate cancer for detection, localization and characterization of primary tumor at the time of initial diagnosis. The two specific aims of this proposal are: 1) to perform a prospective clinical imaging evaluation of 18F-FMAU PET/CT for detection and localization of primary tumor in 40 men with suspected prostate cancer based on elevated/rising prostate specific antigen level including those with prior negative biopsy who are scheduled to undergo clinical mpMRI and TRUS-guided biopsy procedure, and 2) to examine the associations between the PET and mpMRI derived imaging parameters and the biopsy histopathology parameters as well as serum prostate specific antigen level and kinetics. The underlying hypothesis is that 18F-FMAU accumulates in primary prostate cancer allowing for image-directed biopsy with an uptake level that is reflective of tumor proliferation index. We believe that addition of 18F-FMAU PET/CT in conjunction with mpMRI directed prostate biopsy will markedly improve the current status quo and impact the diagnostic evaluation of men with suspected prostate cancer.